There is compelling evidence that tumor-associated hypoxia negatively influences response to chemotherapy and leads to the selection of tumor cells with increased aggressiveness and metastatic potential. Recent studies have identified secreted markers of tumor hypoxia that can be measured in patient plasma. The status of these factors in cancer patients may provide valuable prognostic information and be predictive of response to therapy. In two preliminary studies, we have quantified plasma concentration of three hypoxic markers, plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF) and osteopontin (OPN), demonstrating feasibility and providing sufficient evidence of correlation to warrant testing in larger patient cohorts. We propose to investigate these markers in patient samples from three multi-institution clinical trials to test their predictive and prognostic utility in lung cancer. The ability to accurately predict patient outcome using plasma markers will provide a rapid, minimally-invasive test that can be used to optimize therapy regimens on an individual patient basis. Our central hypothesis is that levels of secreted markers of hypoxia will correlate with response to therapy and overall survival. Aim 1: Determine whether markers of hypoxia correlate with patient outcome under the influence of chemotherapy and radiation, with and without tirapazamine. This Aim will be conducted in two parts. 1A: Investigate pretreatment plasma concentrations of PAI-1, VEGF and OPN, using established ELISA techniques in plasma isolated prior to initiation of treatment. Expression levels will be correlated with patient response, progression-free survival and overall survival using univariate and multivariate statistical analyses. 1B. Investigate protein expression of hypoxic markers in archival tumor tissue to correlate this measure of tumor hypoxia with plasma concentrations of secreted hypoxic markers. We will investigate the expression of six proteins associated with tumor hypoxia in archival specimens as measured by immunohistochemistry. Aim 2: Determine whether treatment-induced changes in plasma concentrations of PAI-1, VEGF and OPN correlate with patient outcome. We will investigate whether increases or decreases in plasma concentration of secreted markers of hypoxia under the influence of therapy correlate with patient response to therapy, progression-free survival and/or overall survival.